EP1782821A1 - Arzneimittel und verfahren zur verbesserung der gehirnfunktion - Google Patents

Arzneimittel und verfahren zur verbesserung der gehirnfunktion Download PDF

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EP1782821A1
EP1782821A1 EP05767287A EP05767287A EP1782821A1 EP 1782821 A1 EP1782821 A1 EP 1782821A1 EP 05767287 A EP05767287 A EP 05767287A EP 05767287 A EP05767287 A EP 05767287A EP 1782821 A1 EP1782821 A1 EP 1782821A1
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Prior art keywords
growth factor
cerebral
hgf
cellular growth
function
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French (fr)
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EP1782821A4 (de
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Yasufumi Kaneda
Ryuichi Morishita
Munehisa Shimamura
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Anges Inc
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Anges MG Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a medicament and a method for amelioration of a cerebral function. More particularly, the present invention relates to a medicament for amelioration of a cerebral function which contains a cellular growth factor or the like as an active ingredient, and a method for amelioration of a cerebral function.
  • the brain is the most important organ among various organs, but it is also the least studied field. Within this field, control of cerebral functions has not been fully explained in relation to its actual mechanisms. In particular, it is unknown if amelioration of physiological conditions of the brain (for example, neoangiogenesis) will directly result in amelioration of cerebral functions in any ways, and there are a number of unknown points with regard to the control of cerebral functions (for example, amelioration, prevention of deterioration and the like).
  • physiological conditions of the brain for example, neoangiogenesis
  • Cerebral occlusive diseases, occlusive disease of the circle of Willis and the like caused by atherosclerosis in the artery of the brain often cause chronic reduction in cerebral blood flow. This state may not only cause subsequent cerebral ischemia but also neuropathological alteration including dementia, that is, decline in cerebral functions ( Sekhon LH, Morgan MK, Spence I, Weber NC., Stroke 25, 1022-1027 (1994) ; Stroke 29, 1058-1062 (1998) ; Stroke 24, 259-264 (1993) ; and Kalaria RN, Bhatti SU, Lust WD, Perry G., Ann. N.Y, Acad. Sci. 695, 190-193 (1993) . However, no effective therapy for the amelioration of such decline in cerebral functions has been established.
  • angiogenic growth factor A growth factor which has been shown to stimulate such neoangiogenesis in vivo and possess mitogenic effect on endothelial cells in vitro is referred to as "angiogenic growth factor”. Involvement of angiogenic growth factors in therapy was published for the first time by Folksman et al. in an article ( Folksman et al., N. Engl. J. Med. 285, 1182-1186 (1971) ).
  • fibroblast growth factor (FGF) family Science257, 1401-1403 (1992) and Nature 362, 844-846 (1993)
  • endothelial cell growth factor J. Surg. Res, 54, 575-583 (1993)
  • VEGF vascular endothelial growth factor
  • HGF also acts as an endothelium-specific growth factor , which is similar to VEGF ( J. Hypertens. 14, 1067-1072 (1996) ).
  • therapeutic angiogenesis Strategy of using an angiogenic growth factor as described above for therapy of vascular disorder is referred to as "therapeutic angiogenesis”. More recently, this strategy has been applied to human ischemic diseases. However, the effectiveness of this strategy for amelioration of the decline in cerebral functions or for the prevention of deterioration thereof is unknown at the present time and is unpredictable from the knowledge obtained so far.
  • HGF hepatocyte growth factor
  • HGF transmembrane tyrosine kinase c-Met/HGF receptor
  • functional binding between HGF and c-Met enhances survival of neurons in a primarily cultured hippocampus
  • neurite outgrowth is induced in the development of neurons in vitro ( J. Cell. Biol. 126, 485-494 (1994) and Japanese Laid-Open Publication No. 7-89869 ).
  • HGF is induced in ischemic neurons ( Brain Res.
  • a recombinant HGF has neuroprotective effects against tardive neuronal death after ischemia in the hippocampus, and that continuous injection of recombinant HGF into a brain is effective for reducing the degree of an infarction ( J. Cereb. Blood Flow Metab. 18, 345-348 (1998) .
  • an HGF can act as an important neurotrophic factor during cerebral ischemia.
  • the effectiveness of HGF for the amelioration of cerebral functions is unknown and is unpredictable.
  • vascular endothelial growth factor is a dimeric glycoprotein which has a mitogenic effect on an endothelial cell, and also has an ability of enhancing vascular permeability.
  • VEGF has direct and specific mitogenic effects on an endothelial cell ( Biochem. Biophys. Res. Commun., 161, 851-858 (1989) ). Binding sites of a VEGF including tyrosine kinase receptor, Flt, Flk-1 and KDR are found on endothelial cells, but not on other types of cells, and thus the effects of a VEGF are limited to endothelial cells.
  • VEGF vascular endothelial growth factor
  • neoangiogenesis is believed to play an important role in the recovery from cerebral ischemia or in the prevention of future paroxysm.
  • neoangiogenesis is related to amelioration of decline in cerebral functions or prevention of deterioration of cerebral functions (for example, amelioration of memory function and amelioration of learning function).
  • cerebral diseases and disorders may be considered as the last area in which only a few solutions have been found.
  • the demand for therapy and prophylaxis of cerebral diseases and disorders for the purpose of amelioration of decline in cerebral functions and prevention of deterioration of cerebral functions have increased year after year.
  • cerebral diseases those caused by atherosclerosis in cerebral arteries, occlusive disease of the circle of Willis and the like often cause chronic cerebral hypoperfusion.
  • vascular endothelial growth factor VEGF
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • the problemof the present invention is to provide a novel therapeutic agent for the amelioration of cerebral functions or the prevention of deterioration of cerebral functions and a novel administration method of such a therapeutic agent.
  • the present invention has solved the above problem by providing a composition containing a growth factor such as HGF (hepatocyte growth factor) gene and/or VEGF (vascular endothelial growth factor) gene as an active ingredient, which has acts as therapy for disorders of cerebral functions or prevention of deterioration of cerebral functions, and enhances neurite outgrowth or synaptogenesis.
  • a growth factor such as HGF (hepatocyte growth factor) gene and/or VEGF (vascular endothelial growth factor) gene
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • the present invention relates to the following items:
  • the present inventors performed in vivo studies testing whether cerebral functions can be ameliorated by administration of a cellular growth factor such as HGF, FGF and VEGF. As a result, the present inventors clarified that administration of a cellular growth factor resulted in significant amelioration of cerebral functions such as memory function and spatial learning function, and enhanced neurite outgrowth or synaptogenesis, in particular with regard to the region of cerebral infarction and therearound.
  • a cellular growth factor such as HGF, FGF and VEGF.
  • the present invention provides a composition for the amelioration of decline in cerebral functions or prevention of deterioration of cerebral functions and enhancement of neurite outgrowth or synaptogenesis, in particular with regard to the region of cerebral infarction or therearound.
  • Cerebral function refers to a complicated activity performed by many parts of a cerebrum. Examples of such a function include language function, cognitive function, motor function, memory function, learning function, attention function and the like.
  • cogntive function refers to ability of an organism to recognize identity of other organisms. “Cognitive function” is confirmed by, for example, behavioral tests, spontaneous activity tests, Morris water maze tests and the like.
  • motor function refers to management of motion by the nerve or brain.
  • Motor function can be confirmed by, for example, behavioral tests, spontaneous activity tests, Morris water maze tests and the like.
  • memory function refers to a function of the brain or nervous system to memorize a previously experienced state or information. “Memory function” can be confirmed by, for example, behavioral tests, Morris water maze tests, passive avoidance learning and the like.
  • learning function refers to a function of the brain or nervous system to memorize and use a previously experienced state. "Learning function” can be confirmed by, for example, behavioral tests, Morris water maze tests, passive avoidance learning and the like.
  • Hepatocyte growth factor used in the present invention is described in, for example, Nature, 342, 440 (1989 ), Japanese Patent Publication No. 2577091 , Biochem. Biophys. Res. Commun., 163, 967 (1989 ), Biochem. Biophys. Res, Commun., 172, 321 (1990 ) and the like. Representative sequences will be set forth in SEQ ID NOS:1 and 2 (nucleic acid sequence and amino acid sequence respectively). To express such an HGF, HGF cDNA can be incorporated into an appropriate expression vector, as will be described below (nonviral vector and viral vector).
  • Base sequences of cDNA encoding an HGF here are also registered in a database such as Genbank, in addition to those described in the literatures described above. Accordingly, based on these sequence information, for example, RT-PCR reaction can be performed with respect to a mRNA derived from a liver or leucocyte using an appropriate DNA portion as a PCR primer, thereby cloning a cDNA of an HGF.
  • RT-PCR reaction can be performed with respect to a mRNA derived from a liver or leucocyte using an appropriate DNA portion as a PCR primer, thereby cloning a cDNA of an HGF.
  • Those skilled in the art would be able to readily perform such cloning in accordance with a basic reference, for example, Molecular Cloning 2nd Edt. , Cold Spring Harbor LaboratoryPress (1989 ) and the like.
  • an HGF of the present invention is not limited to the above HGF. Any protein may be used as an HGF of the present invention, as long as the expressed protein is a protein having substantially the same action as an HGF.
  • an HGF of the present invention can also encompass a protein having substantially the same action as an HGF and consists of an amino acid sequence including one or a plurality of (preferably, several) amino acid substitution, deletion and/or addition with respect to an amino acid sequence of a well-known HGF protein such as a protein encoded by the above cDNA.
  • nucleic acid DNA and RNA
  • form of protein with regard to a cellular growth factor refers to a cellular growth factor itself (that is, a protein) or a protein having substantially the same action as the cellular growth factor.
  • Form of nucleic acid includes a nucleic acid encoding a cellular growth factor itself or a protein having substantially the same action as the cellular growth factor, or a nucleic acid which hybridizes with such a nucleic acid under stringent conditions (herein, these nucleic acids may be represented as a gene), and indicates a form such that, when administered to a subject, a protein encoded by such a nucleic acid is expressed in the subject.
  • a protein encoded by the nucleic acid is expressed in the subject, and the protein attains actions as a cellular growth factor.
  • VEGF Vascular endothelial growth factor
  • VEGF121 vascular endothelial growth factor
  • VEGF165 vascular endothelial growth factor
  • VEGF189 vascular endothelial growth factor
  • VEGF206 vascular endothelial growth factor
  • Fibroblast growth factor (FGF) as used herein is described in, for example, Diilber MS, et al. (1994) Exp Hematol 22(12):1129-33 , and is expressed in various normal cells and cancer cells.
  • FGF basic
  • aFGF acidic
  • FGF3 which is an int-2 gene product
  • FGF4 which is a K-fgf/hst1 gene product
  • FGF5 which is a K-fgf/hst1 gene product
  • FGF5 which are hst2 gene products
  • KGF keratinocyte growth factor
  • AIGF keratinocyte growth factor
  • FGF9 which have similar structure and functions, such as strong binding to heparin.
  • neoangiogenesis refers to the formation of a new blood vessel and an activity of such formation.
  • neoangiogenic action refers to the ability of a factor to form a new blood vessel in a target when acting on the target.
  • neurite outgrowth refers to the outgrowth of any neurite, and is confirmed by observing if substantial outgrowth of a neurite is present using a microscope or the like.
  • synapse refers to the conjugation between neurons or between a neuron and other cells (synaptic connection) or between conjugation sites. Synapse may be observed using an electron microscope.
  • the main features defining a synapse may be (1) the synaptic vesicle, (2) the synaptic cleft, (3) the thickening of presynaptic membrane and subsynaptic membrane (post-synaptic membrane) and the like. Therefore, as used herein, “synaptogenesis” refers to formation of a synapse in a position without synapse. The formation of a synapse can be confirmed by verifying the presence of synapse using the above method.
  • Hemagglutinating virus of Japan and “HVJ” refer to virus belonging to Paramyxoviridae or Paramyxovirus having a cellular fusion action and are interchangeably used.
  • a genome therof is a minus-strand RNA consisting of approximately 15500 bases.
  • Viral particle of Hemagglutinating virus of Japan has an envelope, and is polymorphic with a diameter of 150-300nm.
  • Hemagglutinating virus of Japan has RNA polymerase. This virus is unstable under heat. It aggregates almost every type of erythrocytes and is hemolytic.
  • Hemagglutinating virus of Japan tends to cause persistent infection when it infects an established cell. Since it has the ability of fusing various cells, it is broadly used for cellular fusion such as for the formation of heterokaryon and the preparation of a hybrid cell.
  • (viral) envelope refers to a membrane structure based on a lipid bilayer surrounding a nucleocapsid present in a specific virus such as Hemagglutinating virus of Japan.
  • An envelope is typically found in viruses which mature by budding from a cell.
  • An envelope generally consists of a small projection structure consisting of spike protein encoded by a viral gene and lipids derived from a host. Therefore, "(viral) envelope vector” is a term used in the case where an envelope is used as a vector (carrier), and it may be interchangeably used herein with (viral) envelope in some cases.
  • AAV adeno-associated virus
  • retrovirus Franceschini IA, et al., J Neurosci Res. 2001;65:208-19
  • adenovirus Miyaguchi K, Maeda Y, Colin C, Sihag RK., Brain Res Bull. 2000; 51:195-202
  • herpes simplex virus Johnson PA, Yoshida K, Gage FH, Friedmann T., Brain Res Mol Brain Res.
  • HVJ-E vector is effective in transfecting a gene to the central nervous system without any apparent toxicity. Hence, HVJ-E vector may be used in the present invention.
  • HJV Hemagglutinating Virus of Japan
  • This vector system was developed based on the first-generation HVJ-based gene transfer using a viral envelope and liposome (HVJ liposome method, Yamada K, et al., Am J Physiol. 1996;271:R1212-20 ; Kaneda Y, et al., Exp Cell Res. 1987;173:56-69 ).
  • the first-generation HVJ vector has great potential with regard to transfection to the central nervous system of rat and Primate (Yamada K. et al.
  • HVJ-envelope (HVJ-E) vector which is a novel nonviral vector system, uses only an envelope of HVJ to transfer exogenous gene.
  • inactivation refers to inactivation of a genome, when it is used with regard to virus (for example, Hemagglutinating virus of Japan).
  • Inactivated virus is replication-defective virus.
  • Inactivation is achieved by a method described herein (for example, alkylation and the like). Examples of such a method for inactivation include, but not limited to, a method including the steps of: (a) inactivating virus (for example, HVJ) by treatment with alkylating agent; (b) obtaining concentrate of the virus or inactivated virus; and (c) purifying the virus or inactivated virus by column chromatography and subsequent ultrafiltration, and a method in which the order of these steps are changed.
  • inactivating virus for example, HVJ
  • alkylation refers to substitution of hydrogen atom of an organic compound with an alkyl group.
  • Alkylating agent refers to a compound which provides an alkyl group. Examples of alkylating agent include alkyl halide, dialkyl sulfate, alkyl sulfonate, dialkylzinc. Examples of preferred alkylating agent include, but are not limited to, ⁇ -propiolactone, butyrolactone, methyl iodide, ethyl iodide, propyl iodite, methyl bromide, ethyl bromide, propyl bromide, dimethyl sulfate, diethyl sulfate and the like.
  • the present invention also relates to a method or therapy for the amelioration of cerebral functions or prevention of deterioration of cerebral functions.
  • the present invention relates to a method or therapy for enhancing neurite outgrowth or synaptogenesis (a subject is particularly, but not limited to, those caused in a region of cerebral infarction or therearound).
  • parenteral delivery method includes local administration, intraarterial administration (for example, via carotid artery), intramuscular administration, subcutaneous administration, intramedullary administration, administration to a subarachnoid space, intraventricular administration, intravenous administration, intraperitoneal administration, intranasal administration and the like.
  • any route may be used as long as the route delivers the composition to a section to be treated.
  • gene therapy refers to a method for therapy of diseases by introduction of a nucleic acid (forexample, DNA) to a patient. While gene therapy includes a method using the step of injecting a naked nucleic acid, a vector can be used in many cases. In the present invention, a viral envelope is used as a vector. Gene therapy is performed by administration of an expressed or expressible nucleic acid to a subject. In such an embodiment of the present invention, a nucleic acid produces a protein encoded by the nucleic acid, and such a protein mediates therapeutic effects.
  • any method for gene therapy used in the field of the art may be used in accordance with the present invention. Exemplary methods are described in common general information manuals such as: Goldspie L et al., Clinical Pharmacy 12:488-505 (1993) ; Wu and Wu, Biotherapy 3:87-95 (1991) ; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993) ; Mulligan, Science260:926-932(1993) ; and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993) ; May, TIBTECH 11(5):155-215 (1993) . Generally known DNA recombination techniques are described in: Ausubel et al. (ed.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993 ); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990 ).
  • a composition and a medicament of the present invention may contain, in addition to viral envelope, an appropriate, pharmaceutically acceptable carrier including an excipient or other compounds to enhance processing of the envelope, for preparing a pharmaceutically usable preparation.
  • pharmaceutically acceptable carrier refers to a substance used in the production of a medicament or veterinary medicine and has no harmful influences on active ingredients.
  • examples of such a pharmaceutically acceptable carrier include, but not limited to, an emulsifier, a suspending agent, a solvent, an extender, a buffer, a delivery vehicle, a diluent, an excipient and/or a pharmaceutical adjuvant.
  • a pharmaceutical composition and a medicament of the present invention may be administered in any aseptic and biocompatible pharmaceutical carrier (including, but not limited to, saline, buffered saline solution and water) . Any of these molecules may be administered to a patient independently or in combination with other agents, in a pharmaceutical composition mixed with an adjuvant and/or pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is pharmaceutically inactive.
  • a pharmaceutical preparation for parenteral administration may be an aqueous solution of an active compound.
  • a pharmaceutical composition of the present invention may be formulated in an aqueous solution, preferably a physiologically adaptable buffer solution such as Hanks' solution, Ringer's solution or buffered saline solution.
  • a suspended active compound may contain a liposome.
  • a pharmaceutical composition of the present invention encompasses a composition containing an effective amount of envelope of the present invention for achieving a desired purpose.
  • “Therapeutically effective amount” or “pharmacologically effective amount” is a term sufficiently recognized by those skilled in the art, and refers to an amount of an agent effective for causing a desired pharmacological result. Therefore, a therapeutically effective is an amount sufficient for alleviation of symptoms of a disease to be treated.
  • One useful assay for confirming the effective amount for a predetermined application is to measure the degree of recovery from a disease of interest. An actual dose depends on an individual to which a treatment is applied, and is preferably an amount optimized so that a desired effect may be achieved without causing any significant side effects. Determination of a therapeutically effective amount is sufficiently within the ability of those skilled in the art.
  • a therapeutically effective amount may be initially estimated using a cell culture assay or any suitable animal model. Animal models may also be used for determining the desired range of concentration and administration route. Subsequently, a dose and route useful for administration to a human can be determined using such information.
  • a therapeutically effective amount refers to the amount of envelope to alleviate the degree or state of diseases.
  • Therapeutic effects and toxicity of a compound may be determined based on cell culture or standard pharmaceutical procedures (for example, ED 50 (therapeutically effective dose for 50% of a group) and LD 50 (lethal dose for 50% of a group). The dose ratio between therapeutic effects and toxic effects is the therapeutic index, and is represented by a ratio of ED 50 /LD 50 .
  • Pharmaceutical compositions with a great therapeutic index are preferred. Data obtained from cell culture assays and animal experiments are used for formulating a range of amount of composition for use in a human. Such a dose of a compound is preferably within the range of circulating concentration containing ED 50 without little or no toxicity.
  • Such a dose varies according to the administration form to be used, susceptibility of a patient and the administration route.
  • dose of an envelope is appropriately selected depending on conditions such as the age of the patient, the type of disease, type of envelope to be used, and the like.
  • an envelope vector corresponding to 400-400,000HAU, preferably 1,200-120,000HAU, more preferably 4,000-40,000HAU may be administered to one subject.
  • An amount of exogenous gene contained in the envelope to be administered may be 2-2,000 ⁇ g, preferably 6-600 ⁇ g, more preferably 20-200 ⁇ g, per subject.
  • HAU refers to the activity of virus capable of aggregating 0.5% of chicken erythrocytes. 1HAU corresponds to almost 24,000,000 viral particles ( Okada, Y. et al., Biken Journal 4, 209-213, 1961 ). The above-mentioned amount may be administered once or several times per day.
  • a pharmaceutical composition with prolonged action may be administered every three or four days, every week, once per two weeks or once per month.
  • Guidance with regard to a specific dose and method of delivery is provided in known literatures in the field of the art.
  • compositions and medicament used in a method of the present invention can be readily determined by those skilled in the art by taking into consideration a purpose of use, subject patient (type, severity and the like), age, body weight, sex, case history of the subject, form or type of cytophysiologically active substance, form or type of a cell and the like.
  • Frequency of application of a method of the present invention to a subject (or a patient) can also be determined readily by those skilled in the art by taking into consideration a purpose of use, a disease to be treated (type, severity and the like), age, body weight, sex, case history and therapeutic progress of the patient. Frequency may be, for example, once per day to once per several months (for example, once per week to once per month). It is preferred to administer once per week to once per month while observing progress.
  • compositions and medicament of the present invention may be used for application to a human, they may also be used for other hosts (for example, mammals and the like).
  • a method for delivering a cellular growth factor to the brain includes the steps of: 1) temporarily occluding an artery of the head or cervix; and 2) introducing a cellular growth factor to the brain while the artery of the head or cervix is occluded.
  • a cellular growth factor is effectively (for example, over twice) introduced to the brain.
  • Examples of a method for temporal occlusion include balloon catheter, clipping and the like, and cerebral infarction as a physiological method. Balloon catheter and clipping is preferred.
  • Temporal means time period sufficient for the administration of a cellular growth factor (for example, at least one min., at least five min. or the like). A preferred time period may be 1-120 min.
  • the present invention relates to use in the production of a medicament for the treatment of disorder of cerebral functions.
  • any embodiments described herein may be used as a preferred embodiment of cellular growth factor.
  • content of DNA in a preparation can be appropriately adjusted depending on the disease to be treated, age and body weight of a patient and the like, it is typically 0.0001-100mg, preferably 0.001-10mg, for a DNA of the present invention.
  • Such a preparation is preferably administered once per several days or several months.
  • the present invention has revealed for the first time that cellular growth factors such as HGF, VEGF, FGF and the like ameliorates or prevents disorder of cerebral functions.
  • cellular growth factors such as HGF, VEGF, FGF and the like ameliorates or prevents disorder of cerebral functions.
  • cerebral function particularly, memory functions and the like
  • the present invention has demonstrated amelioration or prevention at the level of function by the introduction of HGF or VEGF.
  • HGF and VEGF are effectively used as an agent for therapy or prevention of various declines or disorders of cerebral functions, such as the decline or disorder of cerebral functions due to cerebral ischemia, reduction in cerebral blood flow and the like.
  • the present invention is effectively used as an agent for therapy or prevention of the decline or disorder of cerebral functions due to cerebrovascular occlusion, cerebral infarction, cerebral thrombosis, cerebral embolism, cerebral apoplexy (including subarachnoid hemorrhage, transient cerebral ischemia, cerebral arteriosclerosis and the like), cerebral hemorrhage, occlusive disease of the circle of Willis, injury in the head, cerebrovascular dementia, Alzheimer's dementia, sequela of cerebral hemorrhage or cerebral infarction, and the like.
  • the present invention can also be used as an agent for therapy or prevention of the decline or disorder of cerebral functions due to neurodegenerative diseases such as Alzheimer's disease, senile dementia of Alzheimer type, amyotrophic lateral sclerosis, Parkinson's disease or the like.
  • neurodegenerative diseases such as Alzheimer's disease, senile dementia of Alzheimer type, amyotrophic lateral sclerosis, Parkinson's disease or the like.
  • HGF, VEGF, FGF and the like can be respectively used independently or in combination. They can also be used with a gene of other vascular endothelial growth factors. Further, it is also possible to use HGF, VEGF, FGF and the like in the form of nucleic acid and proteins and the like in combination. Preferred combination is a combination of HGF in the form of nucleic acid and HGF protein or VEGF in the form of nucleic acid and VEGF protein. Combination of HGF in the form of nucleic acid and HGF protein is more preferable.
  • HGF protein used herein may be prepared by any method, as long as the protein has been purified so that it can be used as a medicament, and a commercially available product (for example, TOYOBO CO., LTD., Code No. HGF-101 and the like) may also be used.
  • the cDNA of HGF obtained by the above cloning is inserted into an appropriate expression vector, and the expression vector is introduced into a host cell to obtain a transformant, thereby obtaining a recombinant HGF protein of interest from the culture supernatant of the transformant (see, for example, Nature, 342,440 (1989 ), Japanese Patent No. 2577091 ).
  • VEGF protein can also be obtained in the same manner.
  • FGF may be those available from Kaken Pharmaceutical Co., Ltd., or may be prepared based on the sequences obtained from GenBank.
  • compositions containing a growth factor like HGF in the form of nucleic acid such as an expression vector containing cDNA of HGF in an expressible form
  • administration form of such a composition is generally divided into two groups: the case of using a nonviral vector; and the case of using a viral vector.
  • a method for preparing and administering such an expression vector is explained in detail in manuals of experimentation and the like ( Experimental Medicine, Supplement, Idenshichiryo no Kisogijutsu (Basic Techniques of Gene Therapy), YODOSHA CO., LTD.
  • the gene of interest can be introduced into a cell or tissue by the following method.
  • Examples of a method for gene introduction into a cell include lipofection, phosphoric acid-calcium coprecipitation, DEAE-dextran method, direct injection of DNA using a minute glass tube and the like.
  • a method for Examples of a method for gene introduction into a tissue include gene transfer using internal type liposome, gene introduction using electrostatic type liposome, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), gene introduction mediated by a receptor, transfer of a DNA molecule with a carrier (metal particle) to a cell using a particle gun, direct introduction of naked-DNA, introduction using positively-charged polymer and the like. Applying any of these methods, a recombinant expression vector can be incorporated into a cell.
  • HVJ-liposome method a DNA is encapsulated in a liposome formed by lipid bilayer, and then the liposome is fused with inactivated Hemagglutinating virus of Japan (HVJ).
  • HVJ-liposome method is characterized such that fusion activity with a cell membrane is higher than that in a conventional liposome method, and hence, is a preferred form of gene introduction.
  • a method for preparing an HVJ-liposome is described in detail in literatures ( Experimental Medicine, Supplement, Idenshichiryo no Kisogijutsu (Basic Techniques of Gene Therapy), YODOSHA CO., LTD.
  • HVJ HVJ
  • Z strain available from ATCC
  • HVJ HVJ
  • other HVJ strains for example, ATCC VR-907, ATCC VR-105 and the like
  • direct injection of naked-DNA is the simplest method of all the above methods, and in view of this, it is a preferred introduction method.
  • any expression vector may be used, as long as it is capable of expressing a gene of interest in vivo.
  • pCAGGS Gene 108, 193-200 (1991)
  • pBK-CMV pcDNA3.1
  • pZeoSV Invitrogen and Stratagene
  • viral vector such as recombinant adenovirus, retrovirus and the like is used in representative methods. More specifically, a gene of interest is introduced into, for example, a DNA virus or RNA virus such as detoxicated retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Hemagglutinating virus of Japan, SV40, immunodeficiency virus (HIV) and the like, and the cell is infected with the virus, thereby introducing the gene into the cell.
  • a DNA virus or RNA virus such as detoxicated retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Hemagglutinating virus of Japan, SV40, immunodeficiency virus (HIV) and the like
  • Examples of a method for introducing a gene therapy agent into a patient include an in vivo method in which a gene therapy agent is introduced directly into a living body, and an ex vivo method in which a certain type of cell is extracted from a human, and a gene therapy agent is introduced into the cell outside the body, followed by returning the cell into the body ( Nikkei Science (Japanese version of Scientific American), April, 1994, pp. 20-45 , The Pharmaceuticals Monthly, 36(1), 23-48 (1994) , Experimental Medicine, Extra, 12(15) (1994 ), Japan Society of Gene Therapy Ed. , Idenshichiryo Kaihatsu Kenkyu Handobukku (Handbook for Developments and Researches of Gene Therapy), NTS (1999 )).
  • the in vivo method is preferred.
  • an appropriate administration site is selected based on the disease to be treated, symptoms and the like.
  • administration to the lateral ventricle, administration to the subarachnoid space or intracisternal administration and the like are also included.
  • administration to the subarachnoid space is an efficient administration method disclosed in the present invention.
  • administration to the subarachnoid space is preferred.
  • various forms of preparation respectively suitable for the above administration form may be taken.
  • an injection containing a gene as an active ingredient such an injection can be prepared by an ordinary method.
  • it can be prepared by dissolving the active ingredient in an appropriate solvent (buffer solution such as PBS, saline, sterilized water and the like), filtration sterilization using a filter or the like, if necessary, and then filling the solution into an aseptic container.
  • buffer solution such as PBS, saline, sterilized water and the like
  • a commonly used carrier or the like may be added to such an injection, if necessary.
  • a liposome such as HVJ-liposome can be in the form of suspensions, frozen formulations, centrifugation-concentrated frozen formulations, and the like.
  • a sustained-release preparation mini pellet formulations or the like
  • a sustained-release preparation to be embedded close to the diseased part, or is continuously and gradually administered to the diseased site using an osmotic pump or the like.
  • the present invention provides a composition for the prevention of deterioration of cerebral functions or amelioration of cerebral functions, which contains a cellular growth factor.
  • a cellular growth factor supports physical or external amelioration of cerebral vasculature and the like, actual amelioration of functions has not been reported.
  • functions of the nervous network can be ameliorated by mere external amelioration (for example, neoangiogenesis), and the relationship between the ameliorations is also unclear. Therefore, even if neoangiogenesis caused by a factor is observed, it is not expected that the neoangiogenesis is effective for amelioration of the functions or prevention of deterioration of the functions until amelioration of the functions is actually demonstrated.
  • the present invention attains significant effects unexpected by those skilled in the art.
  • the present invention achieves effects of amelioration of cerebral functions or prevention of deterioration of cerebral functions, which could not be achieved conventionally.
  • administration of a cellular growth factor such as HGF to the brain attained amelioration of functions in the Morris water maze test.
  • the present invention provides a composition for enhancing neurite outgrowth or synaptogenesis, which contains a cellular growth factor as an active ingredient.
  • a cellular growth factor supports physical or external amelioration of the cerebral vasculature and the like, enhancement of neurite outgrowth or synaptogenesis has not been reported. Further, it is not recognized that functions of the nervous network can be ameliorated by mere external amelioration (for example, neoangiogenesis), and the relationship between the ameliorations is also unclear.
  • neoangiogenesis by a factor is observed, it is not expected for neoangiogenesis to be effective for the amelioration of the functions or prevention of deterioration of the functions until enhancement of neurite outgrowth or synaptogenesis is actually demonstrated. It is thus recognized that the present invention attains significant effects unexpected by those skilled in the art.
  • the present invention achieves effects of enhancement of neurite outgrowth or synaptogenesis, which could not be achieved conventionally.
  • administration of a cellular growth factor such as HGF to the brain enhanced neurite outgrowth or synaptogenesis.
  • a cellular growth factor used in the present invention preferably has action of inducing vascular growth.
  • a cellular growth factor representatively include VEGF, FGF, HGF and the like. More preferably, HGF is used.
  • Such a cellular growth factor may be administered with a viral envelope.
  • an inactivated viral envelope is preferably used.
  • an envelope of RNA virus may be preferably used.
  • such an viral envelope is preferably an envelope of Paramyxoviridae virus, and more preferably, an envelope of HVJ (hemagglutinating virus of Japan). This is because such an envelope achieves efficient gene introduction and thus is more suitable for gene therapy, although restriction to the theory is not intended.
  • HVJ hemagglutinating virus of Japan
  • a cellular growth factor used in the present invention is provided in the form contained in a viral envelope.
  • Cerebral functions of interest in relation to a composition or method of the present invention for which amelioration or prevention of deterioration is intended include cognitive function.
  • representative examples of such a cerebral function include a cerebral function affected by disorder of cognitive function or motor function.
  • Examples of such a disorder include cerebral infarction, disorders caused by cerebral blood flow disorder, cerebral hemorrhage or cerebrovascular disorder which results in cognitive dysfunction or motor dysfunction.
  • cerebral functions of interest of the present invention include memory function and spatial learning function.
  • a function selected from the group consisting of memory function and spatial learning function has been known so far, and thus the superiority the present invention should be noted.
  • amelioration of such a cerebral function may be achieved by the activation of astrocytes, but not limited thereto.
  • a cellular growth factor used in the present invention may be administered in any form, as long as the factor eventually attains the function in the brain, it may be representatively provided in the form of a protein or nucleic acid.
  • a cellular growth factor is administered in the form of nucleic acid, such a form is referred to as a form of gene therapy.
  • composition of the present invention may be administered through any route as long as the composition is delivered to the brain and achieves amelioration of functions or prevention of deterioration of functions, for example, administration to the subarachnoid space or intracisternal administration is preferred.
  • One of the features of the present invention is that effects of ameliorating functions were observed even after a long period (for example, over one day, two days, three days, four days, five days, six days, seven days and the like) from the occurrence of disorder of cerebral functions.
  • a long period for example, over one day, two days, three days, four days, five days, six days, seven days and the like
  • the effects of the present invention should be appreciated in that no urgency such as within several hours from the occurrence of disorder is required.
  • a composition is administered after six days from the development of disorder of cerebral functions (in case where the day of development is counted as day 1, after day 7).
  • the present invention attains effects as a form of gene therapy, even when a cellular growth factor used is in the form of nucleic acid and is administered after six days from the development of disorder of cerebral functions.
  • the present invention provides a method for the prevention of deterioration of a cerebral function or amelioration of a cerebral function, which includes the step of: (A) administering a cellular growth factor to a patient.
  • a patient as a subject of the present invention may be any organism as long as the organism (for example, mammals such as Rodentia, Primates and the like) has cerebral functions (higher functions such as memory function or learning function, for example).
  • a subject is preferably primates including human, and is more preferably a human.
  • the present invention has great significance as it proved for the first time that a cellular growth factor is effective for an application for amelioration of cerebral functions or prevention of deterioration of cerebral functions.
  • the present invention provides a method for enhancing neurite outgrowth or synaptogenesis, which includes the step of: (A) administering a cellular growth factor to an individual for which enhancement of the neurite outgrowth or synaptogenesis is necessary.
  • a patient as a subject of the present invention may be any organism as long as the organism (for example, mammals such as Rodentia, Primates and the like) has a nervous system (system in which neurite outgrowth or synaptogenesis can be observed).
  • a subject is preferably primates including human, and is more preferably a human.
  • the present invention has great significance as it proved for the first time that a cellular growth factor is effective for an application as an enhancement for neurite outgrowth or synaptogenesis in a cerebral infarction region or therearound.
  • a cellular growth factor used in this method of the present invention may take a form of any composition as explained herein.
  • the present invention provides a use of a cellular growth factor in the production of a medicament for the prevention of deterioration of a cerebral function or amelioration of a cerebral function.
  • a cellular growth factor used in this use of the present invention may take a form of any composition explained herein.
  • a cellular growth factor a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), a hepatocyte growth factor (HGF) and the like can be preferably used.
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • a hepatocyte growth factor (HGF) can be used most preferably.
  • the present invention provides a use of a cellular growth factor in the production of a medicament for the enhancement of neurite outgrowth or synaptogenesis.
  • a cellular growth factor used in this use of the present invention may take a form of any composition explained herein.
  • a cellular growth factor a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), a hepatocyte growth factor (HGF) and the like can be preferably used.
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • a hepatocyte growth factor (HGF) can be used most preferably.
  • mice Male Sprague Dawley rats (350-400g; Charles River Japan, Atsugi, Japan) were anesthetized with pentobarbital sodium (50mg/kg, interperitoneally), thereby allowing natural respiration by the rats. Both sides of the carotid artery were exposed by midline incision in the cervix, and could be firmly ligated with a 2-0 silk.
  • phosphatidylserine, phosphatidylcholine and cholesterol can be mixed in a weight ratio of 1:4.8:2.
  • This lipid mixture (10mg) can be precipitated on a side surface of a flask by removing tetrahydrofuran in a rotary evaporator.
  • Dried lipids can be hydrated in 200 ⁇ l of balanced salt solution (BSS; 137 ⁇ M NaCl, 5.4 ⁇ M KCl and 10 ⁇ M Tris-HCl (pH 7.6)) containing an expression vector in which a gene of interest has been inserted.
  • BSS balanced salt solution
  • Liposomes for the control group contains an expression vector in which a gene of interest has not been inserted (BSS, 200 ⁇ l). Liposomes can be prepared by shaking and ultrasonic treatment of the mixture.
  • HVJ Purified HVJ (Z strain) was inactivated immediately before use thereof by UV irradiation for three minutes (110 erg/mm 2 /sec.).
  • the liposome suspension (0.5ml, containing 10mg of lipids) can be mixed with HVJ (10,000 HAU in BSS of a total amount of 4ml). This mixture was incubated at 4°C for five min., and was then incubated at 37°C for 30min. while gently shaken. Free HVJ was removed from the HVJ-liposome by sucrose density-gradient centrifugation. Top layer of the sucrose gradient can be recovered and used. Final concentration of plasmid DNA was equal to 20 ⁇ g/ml as evaluated in accordance with a previous report ( J. Clin. Invest. 93, 1458-1464 (1994) , Am. J. Physiol. 271, R1212-1220 (1996 )). This preparation method is optimized so as to achieve the maximum transfection efficiency.
  • anesthetized rats can be laid on a stereotaxic frame (Narishige Scientific Instrument Laboratory, Tokyo, Japan), thereby exposing the skull.
  • a stereotaxic frame (Narishige Scientific Instrument Laboratory, Tokyo, Japan)
  • stainless steel cannula (30 gauge; Becton Dickinson, Franklin Lakes, New Jersey) having a Teflon (registered trademark) connector (FEP tube, Bioanalytical Systems, West Lafayette, Indiana) can be introduced into the left lateral ventricle in a manner described in Am. J. Physiol. 271, R1212-1220 (1996 ).
  • Stereotaxic coordinate can be determined as follows: 1.3mm behind bregma, 2.1mm lateral to midline and 3.6mm under skull surface.
  • the HVJ-liposome complex was injected into the lateral ventricle (20 ⁇ l). After injection, the injecting cannula can be removed. It was observed that behavioral change such as convulsion of the extremities or abnormal behavior was not observed in any of the animals subjected to injection.
  • each animal was fixed by the head in the decubitus, and the atlantooccipital membranes were exposed by midline incision of the occipital bone.
  • a stainless steel cannula (27 gauge; Becton Dickinson, Franklin Lakes, New Jersey) was introduced to the subarachnoid space. The position of the cannula was confirmed.
  • the HVJ-liposome solution 100 ⁇ l:100 ⁇ g/ml
  • LIDI laser Doppler imager
  • blood flow can be continuously measured and recorded for two postoperative weeks.
  • LDI laser Doppler imager
  • 2mW helium-neon laser is incorporated in order to cause a ray to continuously scan the surface of a tissue in a size of 12 ⁇ 12cm to a depth of 600 ⁇ m.
  • hemocytes moving in a vasculature change the vibration frequency of incident light in accordance with the Doppler theory. Since a photodiode concentrates diffuse light in a reverse direction, original variation in light intensity is converted into voltage variation in a range of 0-10V.
  • Output perfusion value of 0V can be graduated as 0% perfusion, and 10V can be graduated as 100% perfusion.
  • an image showing the distribution of blood flow distinguished by different colors is displayed on a television monitor.
  • the perfusion signal is divided into six different sections which are respectively displayed as different colors. A portion with reduction in blood flow or a portion with no perfusion is represented by dark blue, while a portion with the maximum perfusion is represented by red.
  • perfusion in the brain surface before occlusion, immediately after occlusion, on day 7 and day 14 after occlusion can be recorded.
  • bone window in a size of 12 ⁇ 12 can be formed with an electric drill. Continuous measurement value can be obtained using this bone window.
  • An image distinguished using different colors is recorded, and then analysis is performed by calculating average perfusion value for each rat.
  • the calculated perfusion value can be represented as a ratio of post-treatment (ischemic) brain to pre-treatment (untreated) brain.
  • 25 ⁇ m frozen section of the coronal plane can be produced from each 100 ⁇ m section for X-gal staining. Staining with X-gal allows the identification of a stained neuron expressing ⁇ -galactosidase.
  • ALP alkaline phosphatase staining
  • 25 ⁇ m frozen section of coronal plane can be produced from each 100 ⁇ m section. These sections are incubated with PBS containing 0.3% hydrogen peroxide to decrease intrinsic peroxidase activity, and then incubated with the first antibody or lectin diluted in PBS containing 10% equine serum at a room temperature for 60 min.
  • biotin-added second antibody suitable for the species and subsequently avidin-biotin peroxidase complex (Vectastain ABC kit, PK6100, Vector Laboratories, Burlingame, California) can be added and the solution can be incubated. Binding of the antibodies can be visualized using diaminobenxidine. It is possible to omit the first antibody and perform staining with an unrelated immunoglobulin which is suitable for the type and class of the antibody for use as a negative control for each antibody.
  • CSF 100 ⁇ l obtained from rats before or 7 or 14 days after occlusion in both sides of the carotid artery were used in these experiments.
  • Rat and human HGF was measured by ELISA kit (Institute of Immunology, Tokyo), and human VEGF can also be measured using ELISA kit (R & D systems, Minneapolis, Minnesota).
  • Human HGF can be obtained for use by cloning human HGF cDNA ( Japanese Patent No. 2577091 ) using an ordinary method and by inserting the clone into an expression vector, pcDNA (available from Invitrogen).
  • Human VEGF can be obtained for use by cloning human VEGF165 cDNA ( Science 246, 1306 (1989 )) using an ordinary method and by inserting the clone into an expression vector, pUC-CAGGS.
  • Human recombinant HGF can be prepared for use by transfecting ovarian cell of a Chinese hamster (ATCC) or C-127 cell (ATCC) with a recombinant expression vector obtained by inserting a human HGF cDNA ( Japanese Patent No. 2577091 ) into an expression vector pcDNA (Invitrogen) and then purifying from the culture medium using an ordinary method.
  • Administration of the present invention can be performed based on, but not limited to, the above materials and experimental methods.
  • HVJ-envelope vector was prepared in accordance with a known method ( Kaneda, Y. et al., Mol Ther 6, 219-26 (2002) and Shimamura, M. et al., Biochem Biophys Res Commun 300, 461-71 (2003) ). Briefly, suspended virus (15000 hemag-glutination units) was inactivated by UV irradiation (99mj/cm 2 ), and was mixed with plasmid DNA (400 ⁇ g) and 0.3% Triton-X.
  • HGF expression vector human HGF cDNA (2.2kb) was inserted into an eukaryotic expression plasmid using a cytomegalovirus (CMV) promoter/enhancer ( Koike, H. et al., Faseb J5, 5 (2003 )). Using this promoter/enhancer, reporter genes were expressed in various cell types. The expression can be regarded as constitutive expression.
  • CMV cytomegalovirus
  • These control vectors were expression vector plasmids with the same structure which contained a promoter but did not contain HGF cDNA.
  • the plasmids were purified using QIAGEN plasmid isolating kit (Qiagen, Hilden, Germany). The prepared sequences is set forth in SEQ ID NO:7 (pcDNA3.1(-)HGF) and SEQ ID NO:8 (pVAX1HGF/MGB1).
  • Wistermale rats (270-300g; Charles River Japan, Atsugi, Japan) were used for this study.
  • poly-L-lysine-coated 4-0 nylon was arranged around the MCA source, thereby occluding the right middle cerebral artery ( Nelayev, L. et al., Stroke 27, 1616-22 (1996) ).
  • the animals were anesthetized with halothane (1-3.5% in a mixture of 70% N 2 O and 30% O 2 ) using a facial mask.
  • halothane 1-3.5% in a mixture of 70% N 2 O and 30% O 2
  • the temperature of the rectum and skull was maintained at 37 ⁇ 0.5°C throughout the surgical procedure (Unique Medical, Tokyo, Japan).
  • the right common carotid artery, right external carotid artery and right internal carotid artery were separated through median incision.
  • 4-0 nylon was inserted from the right external carotid artery, and was inserted therein by 20mm.
  • the right external carotid artery was then ligated using 6-0 nylon.
  • in vivo gene transfer was performed by intercisternal injection (Shimamura, M. et al., 2003, supra). Briefly, the rats were anesthetized with ketamine (Sankyo, Japan) and xylazine (Bayer Ltd., Japan). Each of the animals was fixed by the head with their face down, and the atlantooccipital membrane thereof was exposed through the posterior median incision. A stainless cannula (27 gauge; Becton Dickinson) was then introduced into the cisterna magna (subarchnoid space). After removal of 100 ⁇ l of cerebrospinal fluid (CSF), HJV-envelope vector (100 ⁇ l) containing human HGF was injected at a speed of 50 ⁇ l/min. The animals were then left with their heads down for 30 min.
  • CSF cerebrospinal fluid
  • the present inventors used a simple protocol for evaluating sensorimotor dysfunction using the following categories (maximum score: 4) ( Petullo, D. et al., Life Sci 64, 1099-108 (1999) ). Bending of the forelimb: the rats were fixed by the tail on a flat surface. Paralysis of the forelimb was evaluated based on the degree of bending of the left forelimb. Twist of the body: the rats were fixed by the tail on a flat surface. The degree of torsion of the body was studied. Push to a side: the rats were pushed to the left or right side. Rats with right MCA occlusion showed either weakness with respect to the push to the left side or did not show any resistance. Rearrangement of the posterior limb: one of the posterior limbs was removed from the surface. When the limb was removed, rats with right MCA occlusion slowly rearranged or simply did not rearrange the posterior limb at all.
  • Spontaneous activity was measured through open field test for 30 min. using an automated activity box (Muromachi Kikai, Tokyo, Japan).
  • step-through passive avoidance learning was used.
  • An apparatus having a light chamber and a dark chamber (MEDICAL AGENT, Kyoto, Japan) was used.
  • the rats were put into the light chamber, and the door was left open so that the rats could enter the dark chamber. Rats like dark places, and thus have a habit of going toward the dark chamber.
  • the rats were put into the light chamber, and when the rats entered the dark chamber, they were given a shock at 6.0mA. The respective tests were continued until the rats learn to keep out of the dark chamber for 300 sec.
  • the rats were placed in the light chamber one, three, five and seven days after the acquisition trial. The present inventors then evaluated the latent period (300 sec. to the maximum) during which the rats stayed in the light chamber.
  • the rats were killed and transcardial perfusion fixation was performed in ordinary saline and then in 4% formaldehyde.
  • the brain was extracted, fixed, subjected to cryoprotection treatment, and then sliced into 12 ⁇ m or 30 ⁇ m sections in a cryostat. After blocking, the section was incubated in 3% goat serum and anti-MAP2 antibody (1:1000, Sigma-Aldrich, Saint Louis, MO, USA), anti-GFAP antibody (1:1000, Sigma-Aldrich) and anti-Cdc4 antibody 2 (1:500, Santa Cruz, CA, USA). Thereafter, the section was incubated in an anti-murine fluorescent antibody (1:1000 for NAP2 and GFAP and 1:500 for Cdc42, Alexa Flour 488, Molecular Probes, USA).
  • anti-Cdc42 antibody murine monoclonal antibody; Santa Cruz, CA, USA
  • anti-synaptophysin antibody murine monoclonal antibody; Chemicon, Temecula, CA, USA
  • the obtained image was imported in Adobe Photoshop (version 7.0, Adobe Systems, San Jose, CA, USA).
  • the color image was converted into a gray scale image.
  • This gray scale image was imported in Mac SCOPE (version 2.5, MITANI CORPORATION, Fukui, Japan).
  • ROI was determined as a region of cerebral cortex adjacent to an infarction region. The number of pixels with a signal equal to or higher than 25 was counted.
  • the section was washed in Tris-HCl and then incubated in a substrate solution (mixture of AS-BI phosphate (Sigma-Aldrich) and fast red violet LB salt (Sigma-Aldrich)) for 30min.
  • a substrate solution mixture of AS-BI phosphate (Sigma-Aldrich) and fast red violet LB salt (Sigma-Aldrich)
  • MRI magnetic resonance image
  • the present inventors paid attention to the rats of group A in the present study.
  • the present inventors performed Morris water maze (MWM) test, thereby evaluating spatial learning and memory (DeVries, A. C. et al., Supra). Although all of the rats subjected to MCAo showed an increase in the latent period before reaching the goal in the MWM test, the latent period was gradually reduced for HGF-treated rats in the invisible platform test ( Figure 4a ). In order to eliminate the possibility of influence of loss of vision, sensorimotor dysfunction that will have on the results (DeVries, A.C. et al., Supra), the present inventors also performed a visible platform test.
  • the present invention demonstrated that the results from MWM and passive avoidance learning were significantly preferable for the HGF-treated rat on day 56 and that the size and patterns of infarction did not change. Similar to the previous report (Dijkhuzen, R.M. et al., Supra, Zhang, L. et al., J Neurol. Sci. 174, 141-6 (2000) ), sensorimotor dysfunction was spontaneously recovered for the rats subjected to MCAo, and there was no significant difference between the groups. Although some previous studies have showed that neurotrophic factor have useful effects on the recovery from sensorimotor dysfunction within one month after ischemic disorder ( Kawamata, T et al., Proc. Natl. Acad. Sci.
  • HGF and c-Met are upregulated in astrocytes mainly in the peri-infarcted region after 4-28 days of MCAo, and are related to repair of cerebral tissue after ischemia (Nagayama, T. et al.). From these viewpoints, the present inventors paid attention to the histological alteration in the peri-infarcted region.
  • astrocyte was activated on day 14, but was inactivated on day 56. This is similar to a previous study which has shown effects of BDNF on ischemic disorders ( Schabitz, W.R. et al., Stroke 35, 992-7 (2004) ). In other words, the astrocyte was temporarily inactivated during the acute stage of the disorder.
  • exogenous HGF may temporarily activate an astrocyte, enhance the growth of the axon, and/or directly enhance the growth of axon, but such HGF may not cause proliferation of astrocytes to form a scar of astrocyte (Sun, W. et al., Supra).
  • This discussion is supported by increased immunoreactivity to Cdc42, which has positive effects on neurite growth in neuronal cell (O'Kane, E.M. et al., Supra) in the peri-infarcted region.
  • neoangiogenesis Another possible mechanism may be the influence of neoangiogenesis in the peri-infarcted region. Although it is still unknown whether or not the degree of neoangiogenesis is related to functional recovery, a recent report has demonstrated that collateral growth and new capillaries around stroke in the cortex support recovery of perfusion in the ischemic border region and support long-term recovery in rats ( Wei, L. et al. , Stroke 32, 2179-84 (2001) ). Further, it has been reported that some patients who did not experience immediate clinical amelioration by therapy, despite early arterial recanalization, showed delayed clinical amelioration ( Alexandrov, A.V. et al. , Stroke 35, 449-52 (2004) ).
  • Example 2 example of recovery of cerebral function by VEGF
  • Example 2 experimentation of recovery of cerebral function by another cellular growth factor was performed.
  • the experimentation was performed in the same manner as described in Example 1 except that VEGF (SEQ ID NO:4) was used instead of the HGF in Example 1.
  • cDNA of human VEGF165 was cloned by an ordinary method ( Science 246, 1306 (1989 )), and was introduced into an expression vector, pUC-CAGGS.
  • Example 2 Next, experimentation of recovery of cerebral function by yet another cellular growth factor was performed.
  • the experimentation was performed in the same manner as described in Example 1 except that aFGF (SEQ ID NO:6) was used instead of the HGF in Example 1.
  • the FGF is available from Kaken Pharmaceutical Co., Ltd.
  • the present invention has industrial applicability in the pharmaceutical industry or the like where medicaments for amelioration or prevention of deterioration of cerebral functions are produced.

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US20090082263A1 (en) 2009-03-26
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